EP3781588A1 - Biomarqueur sanguin pour troubles gastro-intestinaux éosinophiliques - Google Patents

Biomarqueur sanguin pour troubles gastro-intestinaux éosinophiliques

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Publication number
EP3781588A1
EP3781588A1 EP19787916.6A EP19787916A EP3781588A1 EP 3781588 A1 EP3781588 A1 EP 3781588A1 EP 19787916 A EP19787916 A EP 19787916A EP 3781588 A1 EP3781588 A1 EP 3781588A1
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EP
European Patent Office
Prior art keywords
cells
tslp
therapy
egid
blood
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EP19787916.6A
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German (de)
English (en)
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EP3781588A4 (fr
Inventor
Marc E. Rothenberg
Ting WEN
Yrina ROCHMAN
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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Publication of EP3781588A1 publication Critical patent/EP3781588A1/fr
Publication of EP3781588A4 publication Critical patent/EP3781588A4/fr
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5409—IL-5
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5437—IL-13
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • the disclosure relates to methods for diagnosing, treating, and monitoring eosinophilic gastrointestinal disorders using a blood-based biomarker assay.
  • TSLP cytokine thymic stromal lymphopoietin
  • EGID eosinophilic gastrointestinal disorders
  • EoE eosinophilic esophagitis
  • EG eosinophilic gastritis
  • the present invention is based, in part, on the surprising discovery of a distinct subpopulation of memory CD4+ T helper cells present in the peripheral blood of human patients with active eosinophilic gastrointestinal disorder (EGID) that is responsive to thymic stromal lymphopoietin (TSLP). Moreover, TSLP responsiveness, as determined according to the methods described here, correlates with disease severity, providing the basis for a blood- based assay for the care and management of EGID. Accordingly, the disclosure provides blood-based biomarker assays, and related compositions and methods, for the diagnosis, treatment, and monitoring of EGID in human subjects.
  • EGID active eosinophilic gastrointestinal disorder
  • TSLP thymic stromal lymphopoietin
  • the disclosure provides methods for detecting TSLP-responsive cells in human blood, the methods comprising
  • EGID eosinophilic gastrointestinal disorder
  • the one or more analytes selected from phosphorylated signal transducer and activator of transcription 5 (pSTAT5), interleukin- 5 (IL-5), and interleukin- 13 (IL-13),
  • the detection of the one or more of the analytes indicates TSLP responsive cells in the blood of the human subject.
  • the detection of the one or more analytes is determined to be positive or negative based upon a predetermined threshold.
  • a positive detection of the one or more analytes indicates TSLP responsive cells in the blood of the human subject.
  • the method for detecting TSLP-responsive cells in human blood is for use in a method for diagnosing or monitoring EGID in a human subject in need thereof.
  • the method further comprises determining an amount of the one or more analytes and determining whether the amount is above or below a diagnostic threshold, wherein an amount above the diagnostic threshold indicates an EGID status of active disease in a method for diagnosing, or disease progression in a method of monitoring EGID.
  • the cells are selected from peripheral blood mononuclear cells (PBMC), CD4+ T cells, and memory CD4+ T cells.
  • the method further comprises a step of isolating a fraction of cells from the whole blood enriched for PBMC, CD4+ T cells, or memory CD4+ T cells.
  • the analyte is pSTAT5.
  • the pSTAT5 is detected by a method comprising flow cytometry.
  • the analyte is IL-5 or IL-13 gene or protein expression.
  • the IL-5 or IL-13 gene expression is detected by a method comprising a polymerase chain reaction (PCR), flow cytometry, or a chromatographic technique.
  • the IL-5 or IL-13 protein expression is detected by a method comprising one or more of flow cytometry, immunoassay, and a chromatographic technique.
  • the EGID is eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), or eosinophilic gastroenteritis (EGE).
  • the subject in need is characterized as presenting with clinical features selected from one or more of dysphagia, food impactions, vomiting, abdominal pain, refractory reflux symptoms, failure to thrive in young children, a diagnosis of an atopic allergic disorder, and a family member having an EGID diagnosis.
  • the atopic allergic disorder is selected from asthma, atopic dermatitis, allergic rhinitis and allergic conjunctivitis.
  • the method further comprises a step of administering an EGID therapy to the subject in need.
  • the EGID therapy is selected from proton pump inhibitor therapy, dietary therapy, anti-cytokine therapy, anti-ALOXl5 therapy, anti- TSLP therapy, anti-eosinophil therapy, glucocorticoid therapy, and esophageal dilation.
  • the EGID therapy comprises anti-TSLP therapy.
  • the anti- TSLP therapy is an immunotherapy.
  • the anti-TSLP therapy comprises anti- TSLP monoclonal antibody therapy.
  • the disclosure also provides a rapid assay for detecting a thymic stromal lymphopoietin (TSLP) responsive population of cells in human blood, the method comprising contacting cells in vitro with TSLP, wherein the cells are previously isolated from a sample of whole blood obtained from a human subject in need of treatment for an eosinophilic gastrointestinal disorder (EGID), and detecting phosphorylated signal transducer and activator of
  • TSLP thymic stromal lymphopoietin
  • the assay may be performed within about 1-6 hours, preferably within about 2-4 hours.
  • the rapid assay is performed by a method comprising flow cytometric analysis of pSTAT5.
  • the cells are selected from PBMC, CD4+ T cells, or memory CD4+ T cells, preferably memory CD4+ T cells.
  • the method further comprises isolating PBMC, CD4+ T cells, or memory CD4+ T cells from the sample of whole blood.
  • FIGs. 1A-B A, flow cytometry analysis of CD4 + and CD8 + -gated T cells from blood and biopsies of normal (N), EoE remission (R), and active EoE (A) patients to detect the expression of a panel of cytokine receptor genes.
  • B flow cytometric double plots of
  • FIGs. 2A-C A, schematic of cytokine stimulation of human CD4 + T cells isolated from blood. Cells were stimulated for 20 minutes with the indicated cytokine, TSLP, IL-2, or IL-4 and assayed for cytokine responsiveness. Responsiveness to IL-4 was assayed by detection of phosphorylated STAT6 (pSTAT6) and to IL-2 or TSLP by detection of phosphorylated STAT5 (pSTAT5).
  • pSTAT6 phosphorylated STAT6
  • pSTAT5 phosphorylated STAT5
  • B flow cytometry plots showing (left most panel) separation of naive from memory CD4+ T cells via expression of CD45RO and the percentage of either memory (top four panels) or naive (bottom four panels) CD4+ T cells detected as positive for either pSTAT6 or pSTAT5 under the indicated conditions of no cytokine stimulation (medium) or stimulation with each of TSLP, IL-4, or IL-2.
  • C flow cytometry plots showing the percentage of pSTAT5 and pSTAT6 positive memory CD4+ T cells in blood obtained from healthy donors (Normal) and patients with active EoE (Active EoE) following either no stimulation (medium) or in vitro stimulation with TSLP (TSLP).
  • Fig. 3A-D A, TSLP responsiveness measured as the percentage of pSTAT5 positive memory CD4+ T cells following in vitro stimulation with TSLP of cells obtained from the blood of healthy donors (Normal or NL), EoE patients in remission (Remission), or EoE patients with active disease (EoE or Active) positively correlates with the number of eosinophils per high power field (Eos/HPF) determined from tissue biopsies.
  • Eos/HPF eosinophils per high power field
  • IL-4 a poor correlation is seen with responsiveness to IL-4 as measured by the percentage of pSTAT6 positive (B) or pSTAT5 positive (C) naive or memory CD4+ T cells following in vitro stimulation with IL-4.
  • D no correlation is observed between responsiveness to IL-2 as measured by the percentage of pSTAT5 positive naive or memory CD4+ T cells following in vitro stimulation with IL-2.
  • Fig. 4A-B CD4 + T cells were isolated from healthy subjects (normal, light bars) and patients with active EoE (Active EoE, dark bars) and either left unstimulated (Ctrl) or stimulated with either TSLP, IL-4, or both TSLP and IL-4 for 3 days then analyzed for (A) gene expression of IL-5 (top panel) and IL-13 (bottom panel) using RT-PCR to quantitate mRNA levels, or (B) IL-5 protein secretion using ELISA. Mean +/- SEM.
  • Figs. 5A-B (A) PCR primer design for H3K27Ac-ChIP-PCR. Three sets of TaqMan primers (gray rectangles below arrows) were designed based on ChIP-sequence results and ENCODE data. (B) Purified CD4 + T cells from normal donors were activated in the presence or absence of TSLP or IL-4 for 3 days and subjected to ChIP with anti-H3K27Ac. PCR products were normalized to their inputs (5%). Data represent duplicate experiments. Mean +/- SEM.
  • Figs. 7A-E mRNA expression of A, CRLF2 (TSLPR), B, IL-5, and C, IL-13 in naive and memory CD4+ T cells isolated from the blood of healthy donors (normal) and patients with active EoE (EoE) either without (-) or with (+) TSLP stimulation, B, C.
  • EoE active EoE
  • FIG. 8 Heat map showing individual results of expression profiling of memory CD4 + T cells obtained from 3 EoE patients, in the presence or absence of TSLP. Seventy-two transcripts were induced and fifty-one were downregulated by stimulation with TSLP compared to unstimulated.
  • FIG. 9 Schematic depicting diagnostic assay process flow for rapid detection of TSLP responsive cells isolated from whole blood using a flow cytometry based assay.
  • the inventors’ discovery of TSLP-responsive memory CD4+ T helper cells in the peripheral blood of human patients with active EGID, and the relationship to disease status, provides the basis for improved patient care in the form of a non-invasive blood-based assay for EGID status that can serve as an alternative or complement to endoscopy in the diagnosis and monitoring of EGID in human patients.
  • the methods described here may advantageously form part of a therapeutic regimen effective to reduce the total number of endoscopies required to monitor disease and treat the EGID patient.
  • the number of endoscopic procedures required may be reduced, e.g., from twice yearly to once per year or less.
  • Eosinophilic gastrointestinal disorders are a diverse group of disorders characterized by increased eosinophil counts in one or more parts of the gastrointestinal tract in the absence of known causes for eosinophilia (e.g., secondary infections) or an underlying systemic inflammatory disease such as inflammatory bowel disease.
  • EGID include disorders such as eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), and eosinophilic gastroenteritis (EGE).
  • TSLP is one of the key pro-allergic cytokines associated with EGID, and in the context of EoE, a TSLP-elicited basophil response may contribute to pathogenesis. Noti et al, Nature Med. 2013 19:1005.
  • Our laboratory previously reported elevated TSLP levels in the esophageal tissues of individuals with EoE. (Blanchard C., et al, J. Clin. Invest. 2006 H6(2):536-547) and a strong association of single-nucleotide polymorphisms (SNPs) in the TSLP locus with susceptibility to EoE (Rothenberg ME., et al, Nat. Genet. 2010 42(4):289- 291).
  • SNPs single-nucleotide polymorphisms
  • Isolated human CD4+ T cells can be “pre-activated” in vitro by stimulation with anti-CD3 and anti-CD28 antibodies which mediate TSLP receptor expression and induce cell responsiveness to in vitro stimulation with TSLP. Rochman et al, J. Immunol. 2007. However, prior to the current disclosure, it was not known that there were endogenous TSLP responsive cells present in the peripheral blood, or that these cells could be used as a biomarker of disease status in EGID.
  • Eosinophilic Gastrointestinal Disorders EGID
  • EGID are classified as“allergic disorders” they have distinct pathology compared to other allergic disorders such as asthma, atopic dermatitis, and celiac disease.
  • the pathogenesis of EGID involves an immune/antigen driven Th2 response.
  • CD4 + T helper cells in the affected tissues produce increased amounts of pro-inflammatory Th2 cytokines including IL-4, IL-5, and IL-13, and these cells have been identified as the primary cellular sources of Th2 cytokines in the pathogenesis of EoE.
  • CD4+ T helper cells may also be referred to interchangeably as“CD4+ T cells” or simply as“T helper cells”, or“Th cells”.
  • Th2 cytokine transcripts and proteins have also been identified in EoE biopsy tissues and are implicated as key contributory factors in EGID, as evidenced by rodent EoE models and anti-IL-l3 therapy in humans. Blanchard C., et al, J. Allergy Clin. Immunol.
  • Standard diagnosis of EGID is dependent upon quantitative assessment of eosinophils in the affected tissue, e.g., esophageal tissue for EoE or gastric tissue for EG.
  • eosinophils in the affected tissue e.g., esophageal tissue for EoE or gastric tissue for EG.
  • HPF high-power field
  • Tissue for diagnosis is obtained using an expensive and uncomfortable invasive endoscopy procedure.
  • a patient will need to undergo endoscopy at least twice a year in order to monitor disease progression and determine therapeutc efficacy.
  • the present methods provide an alternative to endoscopy in the diagnosis and management of EGID in the form of a simple blood-based assay.
  • TSLP-responsive cells are detected in a sample of whole blood obtained from a human subject in need of diagnosis, treatment or monitoring for EGID. Responsiveness to TSLP is determined using an in vitro assay in which isolated cells are stimulated with TSLP for a suitable period of time to induce TSLP-receptor mediated signal transduction.
  • the isolated cells may be peripheral blood mononuclear cells (PBMC), CD4+ T cells, or memory CD4+ T cells.
  • the cells are assayed for the activation or expression of one or more TSLP- targeted proteins selected from phosphorylated STAT5 (pSTAT5), interleukin-5 (IL-5), and interleukin- 13 (IE- 13).
  • TSLP- targeted proteins selected from phosphorylated STAT5 (pSTAT5), interleukin-5 (IL-5), and interleukin- 13 (IE- 13).
  • pSTAT5 phosphorylated STAT5
  • IL-5 interleukin-5
  • IE- 13 interleukin- 13
  • the methods comprise the detection of cells positive for pSTAT5, for example using a method suitable for the detection of specific phosphoproteins, including flourescence activated cell sorting, or“FACS”, also referred to as“flow cytometry”, chromatography based techniques, and solid or liquid phase immunoassays.
  • FACS flourescence activated cell sorting
  • Exemplary techniques include Western blotting, affinity chromatography, thin layer chromatography, high pressure liquid chromatography (HPLC), mass spectrophotometry coupled with HPLC (MS-HPLC), enzyme linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). In some embodiments, combinations of these techniques may also be used.
  • pSTAT5 is detected using a flow cytometry based assay, using a labeled anti-phospho-Stat5 antibody comprising a detectable label suitable for detection by FACS analysis, e.g., a fluorescent label such as phycoerythrin (PE) or allophycocyanin (APC).
  • a detectable label suitable for detection by FACS analysis, e.g., a fluorescent label such as phycoerythrin (PE) or allophycocyanin (APC).
  • PE phycoerythrin
  • API allophycocyanin
  • the disclosure provides a rapid flow-cytometry based assay for detecting TSLP-responsive cells in whole blood. The method is graphically depicted in
  • the method can advantageously be performed in about 4-5 hours from the time of blood draw.
  • the method comprises obtaining a sample of whole blood from the subject, isolating a CD4+ T cell fraction from the whole blood, culturing the CD4+ T cells in vitro and stimulating with TSLP to induce TSLP-dependent signal transduction, fixing and staining the stimulated cells, detecting pSTAT5 in the cells using flow cytometry, and determining EGID disease status based upon the percentage of pSTAT5 positive cells detected.
  • the methods described here may also comprise the detection of IL-5 and IL-13 cytokine expression, for example by assaying for the secreted cytokines in the medium of in vitro cultured cells, or by assaying for cytokine gene or protein expression.
  • Protein expression can be determined by various methods, including flow cytometry, chromatography based techniques and solid or liquid phase immunoassays, and combinations thereof, as described above.
  • Gene expression can be determined, for example, using a polymerase chain reaction (PCR) based method, including a reverse transcription PCR reaction (RT-PCR), a flow cytometry based method, including microfluidics assisted fluorescence in situ hybridization (FISH), or a chromatographic technique such as Northern blotting.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription PCR reaction
  • FISH microfluidics assisted fluorescence in situ hybridization
  • chromatographic technique such as Northern blotting.
  • the cells used in the assays for TSLP-induced cytokine activation and expression described here are obtained from a sample of whole blood obtained from a human subject in need of therapy for EGID, or who presents with clinical symptoms consistent with EGID, as described in more detail below.
  • the term“whole blood” refers to a sample of blood containing cells, such as red blood cells (also referred to as erythrocytes), white blood cells, and platelets, as well as plasma, which is the liquid remaining after the cells are removed.
  • whole blood is obtained from a subject and assayed for the presence of TSLP-responsive CD4+ T cells.
  • the CD4+ T cells are memory T cells.
  • the CD4+ T cells are isolated from the peripheral blood mononuclear cell (PBMC) fraction of the whole blood.
  • the assays are performed using the PBMC fraction, without isolating the CD4+ T cells.
  • peripheral blood mononuclear cells PBMC
  • PBMC peripheral blood mononuclear cells
  • T cells, B cells, natural killer cells lymphocytes
  • monocytes monocytes
  • Other white blood cells referred to generally as granulocytes, have multi-lobed nuclei and include neutrophils, basophils, and eosinophils.
  • the methods may comprise a step of obtaining whole blood from a subject, e.g., by phlebotomy.
  • the blood collected is preferably venous blood which may be collected into a suitable container, e.g., a container comprising sodium heparin.
  • the methods may comprise a step of isolating a PBMC fraction from whole blood, for example by a method comprising density centrifugation, e.g., using FicollTM or similar reagent.
  • the PBMC fraction, CD4+ T cells, or memory CD4+ T cells may also be isolated, for example, using a negative depletion or positive selection strategy, either in combination with density centrifugation or without a centrifugation step.
  • negative depletion non-target cells are tagged with an affinity label which allows for their removal via an affinity reagent, such as an affinity column or similar device.
  • labeling for a negative depletion strategy is accomplished using labelled antibodies against a cell surface antigen located on the non-target cells, i.e., the cells which are to be depleted from the sample, thereby leaving a sample enriched in the target cell population.
  • target cells are tagged with an affinity label and then separated from other non-target cells. Examples of suitable labels include magnetic beads and polyhistidine tags.
  • a combination of one or more of density centrifugation, affinity chromatography, and flow cytometry may be used to isolate the PBMC fraction, the CD4+ T cell fraction, and/or the memory CD4+ T cell fraction from whole blood.
  • the methods described here comprise activating isolated cells in vitro with TSLP in order to induce TSLP-dependent signal transduction in the cells.
  • the amount of TSLP used will be in the range of about 25-100 ng/ml in serum free medium.
  • the time of activation with TSLP will vary depending on the nature of the downstream effector molecule to be detected. For example, where pSTAT5 is to be detected, very short time periods of TSLP stimulation are required, since activation of STAT5 via phosphorylation is an early event in TSLP-dependent signal transduction. In other case, longer periods of stimulation may be required, for example where cytokine gene expression is to be determined, or cytokine protein expression.
  • the method comprises stimulating the cells with TSLP, e.g., about 25-100 ng/ml TSLP, for about 10-60 minutes, e.g., about 10, 20, 30, 45, or 60 minutes, preferably about 20 minutes for the detection of pSTAT5.
  • TSLP e.g., about 25-100 ng/ml TSLP
  • the time period for mRNA and protein detections is 3-4 days.
  • the methods described here comprise determining whether the amount of one or more of the analytes, i.e., pSTAT5, IL-5, or IL-13, is above or below a predetermined diagnostic threshold, wherein the amount relative to the diagnostic threshold indicates EGID disease status, for example the absence of EGID, active EGID, or EGID in remission.
  • an amount of analyte above the diagnostic threshold indicates active EGID in a method for diagnosing, or disease progression in a method of monitoring EGID.
  • the analyte may be the number of CD4+T cells positive for one or more of pSTAT5, IL-5 mRNA, or IL-13 mRNA.
  • the analyte may be the amount of a cytokine produced by the CD4+T cells, as measured by mRNA or protein expression, or secreted protein.
  • the diagnostic threshold for each analyte is pre-determined based on the amount of the analyte in representative populations of healthy disease-free subjects and subjects in one or more disease categories, for example active EoE, EoE in remission, and Celiac disease with EoE.
  • the performance of the method is measured using an area under the curve (AUC) receiver operating characteristics (ROC) curve.
  • AUC area under the curve
  • ROC receiver operating characteristics
  • a reduction in one or more of pSTAT5, IL-5 or IL-13 may be indicative of an absence of exposure to food allergens where the therapy comprises an elimination diet, or inhibition of Th2 cytokine production where the therapy comprises glucocorticoid treatment.
  • a human subject in need of treatment for an EGID may include one who has not yet been diagnosed with EGID but who presents with clinical symptoms of EGID.
  • the subject in need presents with one or more clinical features selected from dysphagia, vomiting, food impaction, abdominal pain, refractory reflux symptoms, failure to thrive in young children, a diagnosis of an atopic allergic disorder, and a family member having an EGID diagnosis.
  • the atopic allergic disorder is selected from food allergy, asthma, atopic dermatitis, allergic rhinitis and allergic conjunctivitis.
  • a human subject in need of treatment for an EGID may include one who has been diagnosed with EGID and is in need of disease monitoring, for example in order to evaluate the efficacy of an EGID therapy.
  • the methods may also comprise a step of treating the subject diagnosed with EGID, and/or modifying the therapy of a subject undergoing monitoring, as described in more detail below.
  • the subject may be treated with one or more EGID therapies, including, for example, proton pump inhibitor therapy, dietary therapy, anti-cytokine therapy, anti-ALOXl5 therapy, anti-TSLP therapy, anti-eosinophil therapy, glucocorticoid therapy, and esophageal dilation.
  • EGID therapies including, for example, proton pump inhibitor therapy, dietary therapy, anti-cytokine therapy, anti-ALOXl5 therapy, anti-TSLP therapy, anti-eosinophil therapy, glucocorticoid therapy, and esophageal dilation.
  • the terms“treatment”,“treating”, or“treat” describe the management and care of a human subject for the purpose of combating EGID and may include the administration of a therapeutic agent as well as the administration of a therapy such as a restricted diet, including for example elemental and elimination diets, or a medical procedure such as esophageal dilation, to alleviate one or more symptoms or complications of EGID, such as EoE or EG, or to eliminate one or more symptoms or complications of EGID, thereby treating the EGID.
  • Therapeutic agents may include small molecules, such as proton pump inhibitors and glucocorticoids, or biologic agents, such as therapeutic antibodies or nucleic acids, including interfering RNAs.
  • Proton pump inhibitor (PPI) therapy may include treatment with a PPI such as dexlansoprazole, esomeprazole, lansoprazole, omeprazole, pantoprazole, and rebeprazole.
  • PPI such as dexlansoprazole, esomeprazole, lansoprazole, omeprazole, pantoprazole, and rebeprazole.
  • Dietary therapy may include, for example, elemental and elimination diets.
  • Anti-cytokine therapy may include, for example, a biologic agent targeted to inhibit cytokine signaling by one or more cytokines via their cognate receptors.
  • the anti-cytokine therapy is an anti-T helper type 2 (Th2) therapy.
  • Th2 immune response is generally characterized by the production of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin- 13 (IE- 13).
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • IE- 13 interleukin- 13
  • an anti-Th2 therapy encompasses a therapy targeting one or more of IL-4, IL-5, and IL-13, and/or their receptors in order to inhibit IL-4, IL-5, and/or IL-13 mediated signal transduction.
  • the most common biologies for anti-cytokine therapy are antibodies, preferably monoclonal antibodies, and most preferably fully human or humanized monoclonal antibodies.
  • the anti cytokine therapy is an anti-T helper type 2 (Th2) therapy selected from one or more of a therapy targeting the IL-4 and/or IL-13 signaling pathway, and a therapy targeting the IL-5 signaling pathway.
  • Th2 anti-T helper type 2
  • Interleukin-4 and interleukin- 13 both mediate inflammation through their receptors, with IL-13 also binding to type 2 IL-4 receptors.
  • IL-4 and IL-13 signaling pathways thus overlap and therapies envisioned by the methods described here may target one or both of these signaling pathways.
  • Therapies targeting IL-4 signaling include monoclonal antibodies such as dupilumab, which targets the IL-4 receptor alpha (IL-4Ra).
  • Therapies targeting IL-13 signaling include monoclonal antibodies such as RPC4046 or tralokinumab, both of which target IL-13.
  • Interleukin-5 (IL-5, CD 125) is an eosinophil growth, activation, and survival factor. Humanized anti-IL-5 antibodies have been shown to be effective in treating asthma patients with the severe eosinophilic form of the disease, as discussed in Rothenberg ME., Cell 2016; 165(3): 509. Therapies targeting the IL-5 signaling pathway include, for example, therapies targeting IL-5 and its receptor, also known as CD 125. Such therapies include monoclonal antibodies such as mepolizumab and reslizumab, which target IL-5, and monoclonal antibodies such as benralizumab, which target the IL-5 receptor.
  • Anti-ALOXl5 therapy is therapy directed at suppressing the expression or activity of the ALOX15 gene product, arachidonate l5-lipoxygenase.
  • ALOX15 inhibitors include PD 146176.
  • Anti-TSLP therapy may take the form of immunotherapy, for example using an antibody against TSLP, such as tezepelumab. Corren L, el al, New Engl. J. Med. 2017; 377(l0):936-946.
  • Anti-TSLP therapy or anti-ALOXl5 therapy may also comprise the administration of a single or double stranded ribonucleic acid (RNA) agent that inhibits the expression of the TSLP gene or the ALOX15 gene, for example, by catalyzing the post-transcriptional cleavage of the target mRNA, or by inhibiting transcription or translation of the target mRNA.
  • the RNA agent is a double stranded or single stranded RNA interference-based agent (RNAi).
  • the RNAi agent may be based on a microRNA (miRNA), a short hairpin RNA (shRNA), or a small interfering RNA (siRNA).
  • the RNAi agent comprises a region that is at least partially, and in some embodiments fully, complementary to the target RNA. Although perfect complementarity is not required, the correspondence should be sufficient to enable the RNAi agent, or its cleavage product in the case of double stranded siRNA or RNAi agents comprising cleavable linkers, to direct sequence specific silencing of the target mRNA, e.g., by RNAi-directed cleavage of the target mRNA.
  • Glucocorticoid therapy may comprise, for example, therapy with one or more glucocorticoids selected from fluticasone, prednisone and budesonide.
  • a method described here may be performed on a “subject” which may include any mammal, for example a human, primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig.
  • the subject is a human.
  • patient refers to a human subject.
  • Example 1 A TSLP responsive subpopulation of human memory CD4 + T cells is present in human blood of patients with EGID
  • TSLP receptor (CRLF2/TSLPR) expression was enriched in CD4 + T cells of esophageal tissue and this expression was elevated in CD4+ T cells of tissue from patients with active EoE compared to both normal tissue and tissue from patients in remission, but TSLP receptor expression was not detectable in CD4+ or CD8+ T cells isolated from blood.
  • the flow cytometry double plot shows that TSLP receptor expression in CD45RO + T cells was significantly increased in the tissue biopsies of active EoE patients compared to normal tissue, but this increase was undetectable in CD45RO + T cells isolated from blood (Fig. 1 B ).
  • FIG. 2A shows a schematic representation of the assay in which human CD4 + T cells were purified from whole blood of healthy or EoE donors and then stimulated for 20 min with either TSLP, IL-4, or IL-2, the latter two cytokines serving as positive controls for responsiveness to IL-4 as indicated by pSTAT6 and responsiveness to IL-2, as indicated by pSTAT5.
  • CD45RO to separate the CD4+ T cells into naive (CD45R0 ) and memory (CD45R0 + ) subpopulations.
  • the responsiveness of CD4+ T cells to TSLP was measured as the percentage of pSTAT5 positive cells detected following either no treatment or stimulation with TSLP, IL-4, or IL-2 (Fig.
  • Fig. 2C shows that there was a significant increase in the percentage of TSLP-responsive cells from patients with active EoE compared to those obtained from normal donors.
  • EOS/HPF eosinophils per high power field
  • EoE patients with active EoE, defined as having at least 15 EOS/HPF, exhibited a significant increase in the percentage of pSTAT5 positive cells compared to normal donors.
  • EoE remission defined as having 2-14 EOS/HPF
  • results in EoE remission showed a moderate elevation in percentage of pSTAT5 positive cells, indicating a non-normalized Th compartment in the disease remission state (p ⁇ 0.001, remission vs. EoE).
  • EoE status as measured by EOS/HPF and the responsiveness of memory or naive CD4+ T cells to IL-4 as measured by the percentage of pSTAT6 (Fig. 3B) or pSTAT5 (Fig. 30 positive cells.
  • IL5 and IL13 mRNA Fig. 4A
  • IL-5 protein Fig. 4B
  • Both TSLP and IL-4 induced the expression of IL5 and IL13 mRNA in CD4+ Th cells isolated from patients with active EoE, but not in cells isolated from normal donors.
  • Co- stimulation with both TSLP and IL-4 further increased the expression of these cytokines in the cells from active EoE patients, but only mild expression of these cytokines was detected in cells from healthy donors.
  • IL-5 protein expression was also differentially increased in the CD4+ Th cells isolated from patients with active EoE, compared to those isolated from normal donors.
  • CD4 + Th cells were isolated from normal donors and activated in vitro as described above. Cells were collected and fixed on day 3, followed by chromatin immunoprecipitation (ChIP) coupled with quantitative polymerase chain reaction (PCR) or“ChIP-PCR” analysis for detection of the histone mark H3K27Ac, which indicates areas of active chromatin.
  • ChIP chromatin immunoprecipitation
  • PCR quantitative polymerase chain reaction
  • H3K27Ac downstream of the IL5 gene, in a locus control region in the RAD50 gene (RHS7), and in a hypersensitivity site (HSS) of an IL3 enhancer induced by TSLP (Fig. 5A-B).
  • Example 2 Blood-based diagnostic assay for EGID.
  • CD4 + T cell isolation was used to detect pSTAT5 induced by TSLP.
  • PBMCs which contain variety of cell populations, were stimulated with TSLP for 20 minutes and then subjected to flow cytometry analysis.
  • CD4 + T cells were detected by gating strategy during flow cytometry analysis.
  • purified and unpurified CD4 + T cells blood samples from same donors were used and percent of pSTAT5 positive cells induced by TSLP was compared. Non-significant differences within the group were observed (Fig. 6E-F). Therefore, unpurified CD4 + T cells method could potentially be used. This will potentially further shorten the processing time and reduce the human error generated during the CD4+ purification.
  • CD4 + Th cells were isolated from the blood of normal and EoE patients and sorted using flow cytometry to differentiate between memory (CD45RO + ) and naive (CD45RA ) CD4+ T subpopulations.
  • the expression level of CRLF2/TSLPR mRNA was significantly higher within population of memory cells from EoE individuals compared to naive cells and memory cells from normal donors (Fig.
  • FIG. 7A The cells were then activated for 3 days in the presence or absence of TSLP.
  • FIG. 7B-C memory CD4 + Th cells from patients with active EoE expressed elevated levels of mRNA for each of IL5 (Fig. 7B) and IL13 (Fig. 7C).
  • the increase in cytokine transcript expression was followed by an increase in protein production of cytokines IL-5 and IL-13 (Fig. 7D) and IL-4, but not IFNy (Fig. 7E).
  • TSLP receptor expression is upregulated in CD4+ T cells obtained from the blood of patients with active EoE at both the mRNA and protein levels and that TSLP strongly induces both pSTAT5 and the expression of Th2 cytokines downstream of STAT5 activation, IL-5 and IL-13 in these cells.
  • Example 3 TSLP-induced alterations in T-cell transcriptomes.
  • TSLP is a Th2 inducer in CD4+ T cells in the blood
  • cytokines and other molecules induced by TSLP To characterize the TSLP-induced transcriptomic changes in an EGID disease context, bulk RNAseq analyses of human blood memory CD4+ T cells was performed using blood obtained from three active EoE patients. In brief, the PBMC fraction was isolated from whole blood (5-10 mL) and the memory CD4 + T cell fraction was isolated and activated in vitro as described above, i.e., by exposure to anti-CD3 and anti-CD28 antibodies and IFNy depletion in the absence or presence of TSLP for 3 days. Samples were subjected to bulk RNA sequencing and bioinformatics analyses using GeneSpring software.
  • RNA sequencing are shown in Fig. 8 as a heat map.
  • Samples 1-3 represent memory CD4+T cells obtained from patients with active EoE. The first three rows show gene expression in unstimulated cells (control) and the next three rows show gene expression in cells stimulated with TSLP (TSLP).
  • TSLP TSLP-activated memory CD4 + T cells derived from EoE active donors
  • TSLP TSLP induced 72 genes and downregulated 51 genes compared to controls. Many of the upregulated genes are known to be involved in allergic inflammation, which validates the importance of TSLP in initiation of allergic responses.
  • FIG. 9 A schematic depicting an exemplary embodiment of a rapid assay for detection of TSLP responsive cells isolated from whole blood is shown in Fig. 9.
  • the entire process from obtaining a blood sample from a subject to data output and EoE status determination may be performed in about 4-5 hours, including the time for initial isolation of the PBMC fraction from whole blood before CD4+ T cell purification, a TSLP stimulation time of about 20 min, sample preparation for flow cytometry analysis (fixation, permeabilization, staining), and flow cytometric analysis.
  • CD4 + T cells are isolated from whole blood, stimulated in vitro with TSLP, followed by fixing and permeabilization of the cells and detection of pSTAT5 using fluorescently-labeled anti-pSTAT5 monoclonal antibodies by flow cytometry.
  • the percentage of pSTAT5 positive CD4 + T cells is determined and compared to a previously determined diagnostic cutoff in order to classify the sample a belonging to a group defined by disease status, e.g., a normal healthy subject, an EoE patient in remission, or an patient with active EoE.

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Abstract

L'invention concerne un dosage biologique simple et rapide basé sur le sang, utile dans le diagnostic, le traitement et la surveillance de troubles gastro-intestinaux éosinophiliques, et des compositions et des procédés associés.
EP19787916.6A 2018-04-20 2019-04-18 Biomarqueur sanguin pour troubles gastro-intestinaux éosinophiliques Pending EP3781588A4 (fr)

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